RhtB protein variants and the method of producing O-phosphoserine using the same

ABSTRACT

The present invention relates to an RhtB (homoserine/homoserine lactone export transporter) protein variant having an enhanced ability to export O-phosphoserine (OPS) that is a precursor of L-cysteine, a polynucleotide encoding the protein, a vector comprising the polynucleotide, an OPS-producing microorganism comprising the protein variant, a method of producing O-phosphoserine using the microorganism, and a method for preparing cysteine or its derivatives, which comprises reacting O-phosphoserine, produced by the method above, with a sulfide in the presence of O-phosphoserine sulfhydrylase (OPSS) or a microorganism that expresses OPSS.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a Divisional of U.S. application Ser. No.14/890,422, filed Nov. 10, 2015, now allowed, which is a U.S. nationalphase application of International PCT Patent Application No.PCT/KR2014/004150, which was filed on May 9, 2014, which claims priorityto Korean Patent Application No. 10-2013-0053428, filed May 10, 2013.These applications are incorporated herein by reference in theirentireties.

STATEMENT REGARDING SEQUENCE LISTING

The Sequence Listing associated with this application is provided intext format in lieu of a paper copy, and is hereby incorporated byreference into the specification. The name of the text file containingthe Sequence Listing is HANO_038_02US_ST25.txt. The text file is 11 KB,was created on Aug. 16, 2017, and is being submitted electronically viaEFS-Web.

BACKGROUND OF THE INVENTION Field of the Invention

The present invention relates to an RhtB (homoserine/homoserine lactoneexport transporter) protein variant having an enhanced ability to exportO-phosphoserine (OPS) that is a precursor of L-cysteine, apolynucleotide encoding the protein, a vector comprising thepolynucleotide, an OPS-producing microorganism comprising the proteinvariant, a method of producing O-phosphoserine using the microorganism,and a method for preparing cysteine or its derivatives, which comprisesreacting O-phosphoserine, produced by the OPS-producing method, with asulfide in the presence of O-phosphoserine sulfhydrylase (OPSS) or amicroorganism that expresses OPSS.

Description of the Prior Art

L-cysteine, an amino acid playing an important role in the metabolism ofsulfur in all living organisms, is used not only in the synthesis ofbiological proteins such as hair keratin, glutathione, biotin,methionine, and other sulfur-containing metabolites, but also as aprecursor for biosynthesis of coenzyme A.

Known methods of producing L-cysteine using microorganisms include amethod of biologically converting D,L-ATC to L-cysteine usingmicroorganisms (Ryu O H et al., Process Biochem., 32:201-209, 1997).Another known method is a method of producing L-cysteine by directfermentation using E. coli (EP 0885962B; Wada M and Takagi H, Appl.Microbiol. Biochem., 73:48-54, 2006). Meanwhile, the present inventorsfound an enzyme (O-phosphoserine sulfhydrylase (OPSS)) that synthesizesL-cysteine from O-phosphoserine (OPS) in certain microorganisms. Basedon this finding, the present inventors developed a method of producingcysteine by reacting OPS with the OPSS enzyme by culturing a mutatedmicroorganism to accumulate OPS therein (Korean Patent Laid-OpenPublication No. 10-2012-004111). The needs still exist to produce OPS inexcessive amounts in order to produce cysteine at high yield.Accordingly, the present inventors have made extensive efforts todiscover an appropriate exporter that enables O-phosphoserine producedin an OPS-producing strain to be released from the cells smoothly. Inaddition, based on various kinds of known transporters, the presentinventors screened ydeD encoding O-acetylserine/cysteine efflux protein,yfiK encoding O-acetylserine/cysteine export permease (Franke I, ReschA, Dassler T, Maier T and Bock A, J. Bacteriology, 185: 1161-166, 2003),rhtB encoding homoserine/homoserine lactone efflux protein (Zakataeva NP, Aleshin V V, Tokmakova I L, Troshin P V, Livshits V A FEBS Lett 1999;452(3); 228-32) and the like, and particularly found that theenhancement of RhtB in the OPS-producing strain results in an increasein the concentration of OPS (Korean Patent Laid-Open Publication No.10-2012-0041115). However, for the production of higher yield ofcysteine, the development of a transporter having a higher ability toexport a precursor OPS from the OPS-producing strain is still required.

SUMMARY OF THE INVENTION

The present inventors have made extensive efforts to discover RhtBprotein variants having increased OPS export activity so as to be ableto further increase the production of OPS, and as a result, haveidentified four novel RhtB protein variants having increased OPS exportactivity, and have found that the proteins can export OPS from anOPS-producing strain more effectively, thereby completing the presentinvention.

It is an object of the present invention to provide an RhtB(homoserine/homoserine lactone export transporter) protein varianthaving enhanced O-phosphoserine (OPS) export activity.

Another object of the present invention is to provide a polynucleotideencoding the protein variant and a vector comprising the polynucleotide.

Still another object of the present invention is to provide anOPS-producing microorganism comprising the protein variant.

Still another object of the present invention is to provide a method forproducing OPS, comprising culturing the microorganism.

Still another object of the present invention is to provide a method forproducing cysteine or its derivatives, comprising reactingO-phosphoserine, produced by the above-described method for producingO-phosphoserine, with a sulfide in the presence of O-phosphoserinesulfhydrylase (OPSS) or a microorganism that expresses OPSS.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram showing a method of producing L-cysteineby accumulating O-phosphoserine from the biosynthesis and microbialfermentation of O-phosphoserine and enzymatically converting theaccumulated O-phosphoserine to L-cysteine.

DETAILED DESCRIPTION OF THE INVENTION

Aspect of the present invention include an RhtB (homoserine/homoserinelactone export transporter) protein variant having enhancedO-phosphoserine (OPS) export activity.

As used herein, the term “O-phosphoserine (hereinafter described as“OPS”)” refers to an ester of serine and phosphoric acid, that is acomponent of many proteins. The OPS is a precursor of L-cysteine and canbe converted to cysteine by reaction with a sulfide under the catalyticaction of OPS sulfhydrylase (hereinafter described as “OPSS”).Accordingly, it is an important factor for increasing productivity ofOPS in the production of cysteine, and thus it is required to developtransporters that enable intracellular OPS to be effectively secretedfrom OPS-producing strains.

As used herein, the term “RhtB (homoserine/homoserine lactone exporttransporter) protein” is known as an exporter of thehomoserine/homoserine lactone that is a precursor of threonine.Information about the RhtB protein is available from known databasessuch as the NCBI GenBank. For example, it may be a protein depositedunder the accession number AAT48223 (EG11469), and the amino acidsequence thereof may be set forth in SEQ ID NO: 1.

As used herein, the expression “RhtB (homoserine/homoserine lactoneexport transporter) protein variant having enhanced O-phosphoserineexporting activity” refers to a protein which has enhanced OPS exportactivity compared to wild-type RhtB protein and which comprises amutation in one or more amino acids of the amino acid sequence ofwild-type RhtB protein. Specifically four RhtB protein variants havingenhanced OPS export activity are identified by inducing random mutationsin a polynucleotide coding the RhtB protein. Among the identifiedprotein variants, a protein variant having an amino acid sequence of SEQID NO: 2 was labeled “RhtB m1”; an RhtB protein variant having an aminoacid sequence of SEQ ID NO: 3 was labeled “RhtB m2”; an RhtB proteinvariant having an amino acid sequence of SEQ ID NO: 4 was labeled “RhtBm3”; and an RhtB protein variant having an amino acid sequence of SEQ IDNO: 5 was labeled “RhtB m4”.

The RhtB protein variants of the present invention include not only theproteins having an amino acid sequence as set forth in SEQ ID NO: 2, 3,4 or 5, but also proteins that have an amino acid sequence showing ahomology of at least 70%, specifically at least 80%, more specificallyat least 90%, even more specifically at least 95%, still even morespecifically at least 98%, and most specifically at least 99% to theamino acid sequences of SEQ ID NO: 2, 3, 4 or 5, and have substantiallyenhanced O-phosphoserine export activity compared to a wild-type RhtBprotein. In addition, it is obvious that proteins comprising a deletion,modification, substitution or addition of one or more amino acids of theamino acid sequence of the RhtB protein are also included in the scopeof the present invention, as long as they comprise an amino acidsequence having the above-described homology and have biologicalactivity substantially identical or comparable to the RhtB protein.

As used herein, the term “homology” refers to the percentage of identitybetween two polynucleotide or polypeptide moieties. The correspondencebetween the sequences from one form to another can be determined bytechniques known in the art. For example, homology can be determined bya direct comparison of the sequence information between two polypeptidemolecules or polynucleotide molecules by aligning the sequenceinformation and using readily available computer programs.Alternatively, homology can be determined by hybridization ofpolynucleotides under conditions which form stable duplexes betweenhomologous regions, followed by digestion with single-stranded-specificnuclease, and size determination of the digested fragments.

As used herein, the term “homologous” in all its grammatical forms andspelling variations refers to the relationship between proteins thatpossess a “common evolutionary origin,” including proteins fromsuperfamilies and homologous proteins from different species. Suchproteins (and their encoding genes) have sequence homology, as reflectedby their high degree of sequence similarity. However, in common usageand in the present invention, the term “homologous,” when modified withan adjective such as “very high,” may refer to sequence similarity andnot a common evolutionary origin.

As used herein, the term “sequence similarity” refers to the degree ofidentity or correspondence between nucleic acid or amino acid sequencesof proteins that may or may not share a common evolutionary origin. Inone embodiment, two amino acid sequences are “substantially homologous”or “substantially similar” when at least about 21% (specifically atleast about 50%, and most specifically at least about 75%, 90%, 95%,96%, 97% or 99%) of the polypeptide match over the defined length of theamino acid sequences. Sequences that are substantially homologous can beidentified by comparing the sequences using standard software availablein sequence data banks, or in a Southern hybridization experiment under,for example, stringent conditions as defined for that particular system.Defining appropriate hybridization conditions is within the skill of theart (see, e.g., Sambrook et al., 1989, infra).

Because the RhtB protein variants of the present invention have enhancedOPS export activity compared to a wild-type RhtB protein, the OPSproductivity of an OPS-producing microorganism can be increased byexpressing the RhtB protein variants of the present invention in themicroorganism.

In an example of the present invention, in order to identify RhtBprotein variants having enhanced OPS export activity, the gene encodingthe wild-type RhtB protein was randomly mutated, and the mutated genewas introduced into recombinant E. coli microorganisms having reducedactivity of endogenous phosphoserine phosphatase (hereinafter describedas “SerB”), and colonies showing the removal of growth inhibition underthe medium conditions containing an excessive amount of OPS, therebyidentifying 4 kinds of RhtB protein variants as described above (Example1). Also, an OPS-producing strain, which expresses the RhtB proteinvariant of the present invention and has reduced activity of endogenousSerB, showed an increase in OPS productivity of up to 10% compared to anOPS-producing strain expressing the wild-type RhtB protein, and a strainwhich expresses the RhtB protein variant and has enhanced activities ofSerA and SerC showed an increase in OPS productivity of up to 14%(Example 2). Such results suggest that the RhtB protein variant of thepresent invention in an OPS-producing strain can significantly increasethe OPS productivity of the strain.

A further aspect of the present invention also includes a polynucleotideencoding the RhtB protein variant and a vector comprising thepolynucleotide.

As used herein, the term “polynucleotide” refers to a polymer ofnucleotide units linked to each other by a covalent bond to form achain. The term generally means a DNA or RNA strand having any length.In the present invention, the term means a polynucleotide fragmentencoding the RhtB protein variant.

As used herein, the term “vector” refers to any vehicle for the cloningof and/or transfer of a nucleic acid into a host cell. A vector may be areplicon to which another DNA segment may be attached so as to bringabout the replication of the attached segment. A “replicon” refers toany genetic element (e.g., plasmid, phage, cosmid, chromosome, virus)that functions as an autonomous unit of DNA replication in vivo, i.e.,capable of replication under its own control. The term “vector” mayinclude both viral and nonviral vehicles for introducing the nucleicacid into a host cell in vitro, ex vivo or in vivo. It may also includeminicircle DNAs. For example, the vector may be a plasmid withoutbacterial DNA sequences. The removal of bacterial DNA sequences whichare rich in CpG regions has been shown to decrease transgene expressionsilencing and result in more persistent expression from plasmid DNAvectors (e.g., Ehrhardt, A. et al. (2003) HumGene Ther 10: 215-25; Yet,N. S. (2002) MoI Ther 5: 731-38; Chen, Z. Y. et al. (2004) Gene Ther 11:856-64). The term “vector” may also include transposons (Annu Rev Genet.2003; 37:3-29), or artificial chromosomes. Specifically, vectors such aspACYC177, pACYC184, pCL1920, pECCG117, pUC19, pBR322 and pMW118 may beused and in an example of the present invention, a pCL1920 vector wasused.

A further aspect of the present invention also includes an OPS-producingmicroorganism comprising the RhtB protein variant.

Herein, the RhtB protein, the RhtB protein variant and the OPS are asdescribed above.

The OPS-producing microorganism comprising the RhtB protein variant ofan example of the present invention may effectively secrete OPS comparedto a microorganism comprising the wild-type RhtB protein, and thus itcan be more useful for the production of OPS that is a precursor ofcysteine.

The OPS-producing microorganism comprising the RhtB protein varianthaving enhanced OPS export activity may be a microorganism comprisingthe RhtB protein variant, which has enhanced OPS export activity due toa mutation in the chromosomal gene encoding the RhtB protein, and/or amicroorganism which comprises the RhtB protein variant having enhancedOPS export activity and is obtained by introducing a vector comprising apolynucleotide encoding the RhtB protein variant, but the scope of thepresent invention is not limited thereto. In an example of the presentinvention, an OPS-producing microorganism comprising a representativeRhtB protein variant having enhanced OPS export activity may beconstructed by introducing into E. coli a vector comprising apolynucleotide encoding the RhtB protein variant having enhanced OPSexport activity.

Further, the OPS-producing microorganism comprising the RhtB proteinvariant may have enhanced activity of the RhtB protein variant.

Methods for enhancing the activity of the RhtB protein include, but arenot limited to, a method of increasing the intracellular copy number ofa gene encoding the protein variant, a method of introducing a mutationinto an expression regulatory sequence for the chromosomal gene encodingthe protein variant, a method of replacing the expression regulatorysequence for the chromosomal gene encoding the protein variant with asequence having strong activity, a method of substituting thechromosomal gene encoding the protein with a gene mutated to increasethe activity of the protein variant, and a method of introducing amutation into the chromosomal gene encoding the protein to enhance theactivity of the protein. The method of enhancing the activity of theprotein can likewise be applied to enhance the activities of otherproteins.

As used herein, the team “introduction” refers to a method oftransferring a vector, which comprises a polynucleotide encoding theRhtB protein variant, to a host cell. This introduction may be easilyperformed using any conventional method known in the art. In general,examples of the introduction method may include CaCl₂ precipitation, theHanahan method that is an improved CaCl₂ method that uses DMSO (dimethylsulfoxide) as a reducing material to increase efficiency,electroporation, calcium phosphate precipitation, protoplast fusion,agitation using silicon carbide fiber, Agrobacterium-mediatedtransformation, PEG-mediated transformation, dextran sulfate-mediatedtransformation, lipofectamine-mediated transformation, anddesiccation/inhibition-mediated transformation. The method fortransforming the vector is not limited to the above-described examples,and any conventional transformation or transfection methods known in theart may be used without limitation.

As used herein, the term “OPS-producing microorganism” refers to aprokaryotic or eukaryotic microbial strain capable of producing OPStherein. For example, the OPS-producing microorganism may be amicroorganism capable of accumulating OPS therein by geneticengineering, but is not limited thereto. For the purpose of the presentinvention, the microorganism may be any prokaryotic or eukaryoticmicroorganism that comprises the RhtB protein variant, and thus canproduce OPS. Examples of the microorganism include microbial strainsbelonging to the genus Escherichia, the genus Erwinia, the genusSerratia, the genus Providencia, the genus Corynebacterium and the genusBrevibacterium. Specifically, the microorganism may be a microorganismof the genus Escherichia. More specifically, it may be E. coli.Particularly, a microorganism of the Escherichia or the genusCorynebacterium can produce OPS and L-serine, because it contains SerA,SerC and SerB proteins that are enzymes in the biosynthesis pathway ofL-serine (Ahmed Zahoor, Computational and structural biotechnologyjournal, vol 3, 2012 October; Wendisch V F et al., Curr Opin Microbiol.2006 June; 9(3):268-74; Peters-Wendisch P et al., Appl EnvironMicrobiol. 2005 November; 71(11):7139-44).

The OPS-producing microorganism may be specifically a microorganismmutated to reduce the activity of endogenous phosphoserine phosphatase(SerB). The SerB has an activity of converting OPS to L-serine, and thusthe microorganism mutated to reduce the SerB activity may have theproperty of accumulating OPS therein, suggesting that it is useful forthe production of OPS. The reduction in the activity of SerB means thatthe activity of SerB is reduced or is removed compared to that in anon-mutated strain. The reduction in the activity of SerB can beachieved using various methods well known in the art. Examples of themethod for reducing the activity of the SerB enzyme include, but are notlimited to, a method of substituting the chromosomal gene encoding theenzyme with a gene mutated to reduce or remove the activity of theenzyme, a method of introducing a mutation into an expression regulatorysequence for the chromosomal gene encoding the enzyme, a method ofreplacing an expression regulatory sequence for the chromosomal geneencoding the enzyme with a gene having weak activity, deleting thechromosomal gene encoding the enzyme, a method of introducing anantisense oligonucleotide that binds complementarily to the transcriptof the chromosomal gene to inhibit the translation of the mRNA into theprotein, a method of artificially adding a sequence complementary to theSD sequence of the gene encoding the enzyme in the front of the SDsequence to form a secondary structure that makes the adhesion ofribosome impossible, and a reverse transcription engineering (RTE)method of adding a promoter to the 3′ end of the open reading frame(ORF) of the corresponding sequence so as to be reverse-transcribed. Inan example of the present invention, using CA07-0012 (accession number:KCCM11121P) disclosed in Korean Patent Laid-Open Publication No.10-2012-004115 and US Patent Laid-Open Publication No. 2012-0190081 as amicroorganism mutated to reduce the activity of endogenous SerB, avector comprising a polynucleotide encoding the RhtB protein variant ofthe present invention was introduced into the microorganism.

Further, the OPS-producing microorganism may be a microorganism havingenhanced activity of phosphoglycerate dehydrogenase (SerA) orphosphoserine aminotransferase (SerC).

The SerA is a protein having an activity of converting3-phosphoglycerate to 3-phosphohydroxypyruvate, and the SerA may be awild-type protein or a mutant resistant to serine feedback inhibition.Also, the SerC is a protein having an activity of converting3-phosphoglycerate to O-phosphoserine. Thus, the microorganism withenhanced activity of SerA and/or SerC may be useful as an OPS-producingstrain. In an example of the present invention, usingCA07-0022/pCL-Prmf-serA*(G336V)-serC (accession number: KCCM11103P),which is a microorganism having enhanced activity of SerA (resistant toserine feedback inhibition) and SerC as disclosed in Korean PatentLaid-Open Publication No. 10-2012-004115, as an OPS-producingmicroorganism, the RhtB protein variant of the present invention wasintroduced into the microorganism to produce OPS. Also, in the presentinvention, the OPS-producing strainCA07-0022/pCL-Prmf-serA*(G336V)-serC-rhtB m1 comprising the RhtB m1protein variant of the present invention, which is a representativestrain having introduced therein the rhtB protein variant havingenhanced OPS export activity, was named “Escherichia coli CA07-0227” anddeposited with the Korean Culture Center of Microorganisms, recognizedas an international depositary authority under the Budapest Treaty, onMar. 7, 2013 under the accession number KCCM11398P (Example 2).

In addition, the microorganism may further have a reduced ability toperform the intracellular uptake or degradation of OPS.

Specifically, the microorganism may be a microorganism mutated to reducethe activity of PhnC/PhnD/PhnE alkylphosphonate ABC transporter (PhnCDEoperon, that is, ATP-binding component of phosphonate transport (PhnC;EG 10713)-periplasmic binding protein component of Pn transporter (PhnD;EG 10714)-integral membrane component of the alkylphosphonate ABCtransporter (PhnE; EG 11283)), alkaline phosphatase (PhoA) or acidphosphatase (AphA).

The OPS-producing microorganism of the present invention may furtherhave enhanced activity of pyrimidine nucleotide transhydrogenase (PntAB;EC 1.6.1.1). As previously described in Sauer U P et al., J Biol Chem.20; 279(8):6613-9. Epub 2003, PntAB participates in the metabolism ofNADPH to regulate the intracellular redox balance.

A further aspect of the present invention also includes a method forproducing OPS, comprising culturing the OPS-producing microorganism.

Herein, the OPS and the OPS-producing microorganism are as describedabove.

Specifically, the method for producing OPS may comprise the steps of: a)culturing an OPS-producing microorganism comprising the RhtB proteinvariant to produce OPS; and b) isolating OPS from the culture of themicroorganism. Specifically, the method may comprise the steps of: a)culturing an OPS-producing microorganism comprising the RhtB proteinvariant to produce OPS; and b) isolating OPS from the culture of themicroorganism, but is not limited thereto.

The OPS-producing microorganism is specifically a microorganism that hasreduced activity of endogenous SerB so as to be able to accumulate OPStherein. In addition, the OPS-producing microorganism may further haveenhanced activity of SerA resistant to serine feedback inhibition and/orSerC, and this activity is as described above.

As used herein, the term “culturing” means growing the microorganismunder artificially controlled conditions. A culturing process in thepresent invention may be performed using a suitable medium and cultureconditions well known in the art. Any person skilled in the art canreadily control the culture process depending on the type of strainselected. Specifically, the culturing may be batch-type culture,continuous culture or fed-batch culture, but is not limited thereto.

In culture of the recombinant microorganism having reduced activity ofendogenous SerB, the medium should additionally contain glycine orserine, because the serine auxotrophy of the recombinant microorganismis induced. Glycine may be provided in the form of purified glycine, aglycine-containing yeast extract, or tryptone. The concentration ofglycine in the medium is generally 0.1-10 g/L, and specifically 0.5-3g/L. In addition, serine may be provided in the form of purified serine,a serine-containing yeast extract or tryptone. The concentration ofserine in the medium is generally 0.1-5 g/L, and specifically 0.1-1 g/L.

In addition, the medium may contain a carbon source. Examples of thecarbon source may include saccharides and carbohydrates such as glucose,sucrose, lactose, fructose, maltose, starch and cellulose, oils and fatssuch as soybean oil, sunflower oil, castor oil and coconut oil, fattyacids such as palmitic acid, stearic acid and linoleic acid, alcoholssuch as glycerol and ethanol, and organic acids such as acetic acid.These carbon sources may be used alone or in combination in the medium.Examples of a nitrogen source that may be contained in the mediuminclude organic nitrogen sources such as peptone, yeast extract, meatjuice, malt extract, corn steep liquor, soybean, and wheat protein, andinorganic nitrogen sources such as urea, ammonium sulfate, ammoniumchloride, ammonium phosphate, ammonium carbonate and ammonium nitrate.These nitrogen sources may be used alone or in combination. Examples ofa phosphorous source that may be contained in the medium includepotassium dihydrogen phosphate, potassium phosphate, and correspondingsodium salts. In addition, the medium may contain metal salts such asmagnesium sulfate or iron sulfate. Additionally, the medium may alsocontain amino acids, vitamins and suitable precursors. These sources orprecursors may be added to the medium in a batch or continuous manner.

Compounds such as ammonium hydroxide, potassium hydroxide, ammonia,phosphoric acid and sulfuric acid may be added to the medium in asuitable manner during culturing to adjust the pH of the culture medium.In addition, during culturing, a defoaming agent such as fatty acidpolyglycol ester may be used to suppress the formation of foam. Further,in order to maintain the culture medium in an aerobic state, oxygen oroxygen-containing gas can be injected into the culture medium. For ananaerobic or microaerobic condition, nitrogen, hydrogen, or carbondioxide may be provided without aeration. The culture medium may betypically maintained at a temperature ranging from 27° C. to 37° C., andspecifically from 30° C. to 35° C. As for the culture period, culturecan be continued until desired amounts of useful substances areproduced. Specifically, the culture period may be 10-100 hours.

In the present invention, the OPS produced in the culturing step mayfurther be isolated and purified. For example, the desired OPS can becollected from the culture using a suitable method known in the artdepending on a culture method, for example, a batch-type culture,continuous culture or fed-batch culture method.

In an example of the present invention, a plasmid comprising each of thefour RhtB protein variants was introduced into an OPS-producing strain,and then the strain was cultured and the OPS productivity thereof wasexamined. As a result, the OPS-producing strain introduced with theplasmid for each of the four protein variants showed an increase in OPSproductivity of up to 14% compared to an OPS-producing strain introduceda plasmid with wild-type RhtB (Example 2).

A further aspect of the present invention also includes a method forproducing cysteine or its derivatives, the method comprising reactingOPS, produced by the above-described OPS production method, with asulfide in the presence of 0-phosphoserine sulfhydrylase (OPSS) or amicroorganism that expresses OPSS.

Specifically, the method for producing cysteine or its derivativescomprises the steps of: a) producing OPS by culturing an OPS-producingmicroorganism comprising the RhtB protein variant; and b) reacting theOPS, produced in step a), with a sulfide in the presence ofO-phosphoserine sulfhydrylase (OPSS) or a microorganism that expressesOPSS, but is not limited thereto. FIG. 1 shows a schematic diagramshowing the process for synthesis of cysteine.

Step a) of the method is as described above. In addition, the method ofthe present invention comprises step b) of reacting the OPS, produced instep a), with a sulfide in the presence of 0-phosphoserine sulfhydrylase(OPSS) or a microorganism that expresses OPSS.

The sulfide that is used in the present invention may be any sulfidethat may be provided not only in a solid form that is generally used inthe art, but also in a liquid or gas form due to the difference in pH,pressure and/or solubility, and may be converted to a thiol (SH) groupin the form of, for example, sulfide (S²⁻) or thiosulfate (S₂O₃ ²⁻).Specifically, the sulfide that is used in the present invention may beNa₂S, NaSH, H₂S, (NH₄)₂S, NaSH or Na₂S₂O₃, which can provide a thiolgroup to OPS. In the reaction, a single thiol group is supplied to asingle reactive OPS group to produce a single cysteine or a derivativethereof. In this reaction, a sulfide is specifically added in an amountof 0.1-3 moles, and specifically 1-2 moles per mole of OPS. Mostspecifically, OPS and a sulfide that provides a thiol group are used ata molar ratio of 1:1 in light of economy.

As used herein, the term “O-phosphoserine sulfhydrylase (OPSS)” refersto an enzyme that catalyzes a reaction in which a thiol (SH) group isprovided to OPS to convert OPS in cysteine. The enzyme was first foundin Aeropyrum pernix, Mycobacterium tuberculosis, Mycobacteriumsmegmatics, and Trichomonas vaginalis (Mino K and Ishikawa K, FEBSletters, 551: 133-138, 2003; Burns K E et al., J. Am. Chem. Soc., 127:11602-11603, 2005). In addition, the scope of OPSS includes not only awild-type OPSS protein, but also a protein variant that comprises adeletion, substitution or addition in one or more nucleotides of apolynucleotide sequence encoding the OPSS and shows activity that isequal to or higher than the biological activity of wild-type OPSSprotein. Further, the scope of OPSS includes the OPSS protein disclosedin Korean Patent Laid-Open Publication No. 10-2012-0041115 and KoreanPatent Registration No. 10-1208267, and its protein variants.

In addition, the method of the present invention may further comprise astep of isolating and purifying the cysteine produced by the reaction ofstep b). Herein, the desired cysteine can be collected by isolating andpurifying it from the reaction solution using a suitable reaction knownin the art.

Further, the cysteine produced by the method of the present inventioncan be easily synthesized into a cysteine derivative by a chemicalsynthesis reaction known in the art.

As used herein, the term “derivatives” refers to similar compoundsobtained by chemically modifying a portion of any compound. Usually, theterm means compounds in which a hydrogen atom or an atom groupsubstituted with another hydrogen atom or atom group.

As used herein, the term “cysteine derivatives” refers to compounds inwhich a hydrogen atom or atom group in cysteine is substituted withanother atom or atom group. For example, the cysteine derivatives mayhave a form in which the nitrogen atom of the amine group (—NH₂) or thesulfur atom of the thiol group (—SH) in cysteine has another atom oratom group attached thereto. Examples of cysteine derivatives include,but are not limited to, NAC (N-acetylcysteine), SCMC(S-carboxymetylcysteine), BOC-CYS(ME)-OH,(R)—S-(2-amino-2-carboxyethyl)-L-homocysteine,(R)-2-amino-3-sulfopropionic acid, D-2-amino-4-(ethylthio)butyric acid,3-sulfino-L-alanine, Fmoc-cys(Boc-methyl)-OH, seleno-L-cystine,S-(2-thiazolyl)-L-cysteine, S-(2-thienyl)-L-cysteine,S-(4-tolyl)-L-cysteine, etc. Cysteine can be easily synthesized into NAC(N-acetylcysteine) by reaction with an acetylation agent, and in basicconditions, it can be synthesized into SCMC (S-carboxymetylcysteine) byreaction with haloacetic acid. These cysteine derivatives are usedmainly as pharmaceutical materials, including cough remedies,cough-relieving agents, and therapeutic agents for bronchitis, bronchialasthma and sore throat.

Hereinafter, the present invention will be described in further detailwith reference to examples. It is to be understood, however, that theseexamples are for illustrative purposes only and are not intended tolimit the scope of the present invention.

Example 1: Identification of RhtB (Homoserine/Homoserine Lactone ExportTransporter) Protein Variants

In order to increase the specificity of an O-phosphoserine (OPS)exporter to increase the OPS-secreting ability of an OPS-producingstrain, the present inventors constructed RhtB protein variants, whichare OPS exporters, in the following manner.

Specifically, to construct protein variants, the rhtB open reading framewas amplified by random mutagenesis PCR (JENA error-prone PCR) using thegenomic DNA of Escherichia coli K12_W3110, ATCC 27325) as a template anda gene-specific primer pair. Each of the gene fragments obtained by thePCR was cleaved with EcoRV and HindIII and cloned into a pCL Prmf vectorcomprising an rmf promoter inserted into a pCL1920 vector (GenBank NoAB236930). Herein, the amplification of the rhtB gene was performedusing primers of SEQ ID NOS: 8 and 9.

The recombinant plasmid libraries constructed by the above-describedprocess were subjected to high-throughput screening (HTS). A platformstrain for the screening was a recombinant microorganism mutated toreduce the activity of endogenous phosphoserine phosphatase (SerB) inthe wild-type E. coli strain W3110 and was named “CA07-0012”(KCCM11212P; Korean Patent Laid-Open Publication No. 10-2012-0041115).

To identify mutants having increased OPS export activity, theconstructed plasmid libraries were transformed into CA07-0012 byelectroporation, and then colonies showing the removal of growthinhibition under medium conditions containing an excessive amount of OPSwere selected. Plasmids were obtained from the selected colonies, andthe nucleotide sequences thereof were analyzed by a sequencingtechnique.

As a result, the following four RhtB protein variants involved inremoving growth inhibition under medium conditions containing anexcessive amount of OPS were selected: an RhtB protein variant having anamino acid sequence of SEQ ID NO: 2 and named “RhtB m1”; an RhtB proteinvariant having an amino acid sequence of SEQ ID NO: 3 and named “RhtBm2”; an RhtB protein variant having an amino acid sequence of SEQ ID NO:4 and named “RhtB m3”; and an RhtB protein variant having an amino acidsequence of SEQ ID NO: 5 and named “RhtB m4”.

Example 2: Examination of OPS Export Activities of rhtB Protein Variantsin OPS-Producing Strain

A plasmid comprising each of the four protein variants identified inExample 1 was introduced into the OPS-producing strain CA07-0012, andthen the O-phosphoserine productivities of the resulting strains wereevaluated.

Specifically, each of the strains was plated on LB solid medium andcultured overnight in an incubator at 33° C. Each of the strainscultured overnight on the LB solid medium was inoculated into a 25-mLtiter medium shown in Table 1 below, and was then incubated in ancultured at a temperature of 34.5° C. and 200 rpm for 48 hours. Theresults of the culture are shown in Table 2 below.

TABLE 1 Composition Concentration (per liter) Glucose 50 g KH₂PO₄ 6 g(NH₄)₂SO₄ 17 g MgSO₄•7H₂O 1 g FeSO₄•7H₂O 5 mg MnSO₄•4H₂O 10 mg L-glycine2.5 g Yeast extract 3 g Calcium phosphate 30 g pH 6.8

TABLE 2 Consumption of glucose O-phosphoserine Name of strain OD562 nm(g/L) (g/L) CA07-0012/pCL- 40 35 1.5 Prmf-rhtB(wt) CA07-0012/pCL- 37 351.7 Prmf-rhtB m1 CA07-0012/pCL- 41 34 1.8 Prmf-rhtB m2 CA07-0012/pCL- 3835 1.8 Prmf-rhtB m3 CA07-0012/pCL- 37 35 1.8 Prmf-rhtB m4

As can be seen in Table 2 above, the strains with the rhtB proteinvariants of the present invention showed excellent results correspondingto an increase in OPS production of up to 10% compared to a strainintroduced with wild-type rhtB gene.

In order to verify the activities of the rhtB protein variants of thepresent invention, the strain CA07-0022/pCL-Prmf-serA*(G336V)-serf(KCCM11103P; Korean Patent Laid-Open Publication No. 10-2012-0041115)having enhanced activities of SerA (D-3-phosphoglycerate dehydrogenase)and SerC (3-phosphoserine aminotransferase), which are involved in thebiosynthesis pathway of OPS, was used as an OPS-producing strain havingan increased ability to produce OPS. To construct apCL-Prmf-serA(G336V)-serC_Prmf-genes vector, the pCL-PrhtB-genes vectorwas amplified using primers of SEQ ID NOS: 6 and 7.

Specifically, each of the strains was plated on LB solid medium andcultured overnight in an incubator at 33° C. Each of the strainscultured overnight on the LB solid medium was inoculated into a 25-mLtiter medium shown in Table 1 below, and was then incubated in ancultured at a temperature of 34.5° C. and 200 rpm for 48 hours. Theresults of the culture are shown in Table 3 below.

TABLE 3 Consumption of glucose O-phosphoserine Name of strain OD562nm(g/L) (g/L) CA07-0022/pCL Prmf 43 33 2.5 serA*C-Prmf-rhtB(wt)CA07-0022/pCL Prmf 41 31 2.6 serA*C-Prmf-rhtB m1 CA07-0022/pCL Prmf 4231 2.6 serA*C-Prmf-rhtB m2 CA07-0022/pCL Prmf 42 32 3.0 serA*C-Prmf-rhtBm3 CA07-0022/pCL Prmf 41 31 2.6 serA*C-Prmf-rhtB m4

As can be seen in Table 3 above, when the rhtB protein variants of thepresent invention were introduced into the OPS-producing strain havingan enhanced ability to produce OPS, the production of OPS in the strainwas increased by up to 14%, suggesting that the rhtB protein variants ofthe present invention are useful for the production of OPS.

In addition, the strain CA07-0022/pCL Prmf serA*C-Prmf-rhtB m1 that is atypical strain introduced with the rhtB protein variant having enhancedOPS export activity was named “Escherichia coli CA07-0227” and depositedwith the Korean Culture Center of Microorganisms, recognized as aninternational depositary authority under the Budapest Treaty, on Mar. 7,2013 under the accession number KCCM11398P.

Although the preferred embodiments of the present invention have beendescribed for illustrative purposes, those skilled in the art willappreciate that various modifications, additions and substitutions arepossible, without departing from the scope and spirit of the inventionas disclosed in the accompanying claims.

What is claimed is:
 1. A method for producing O-phosphoserine (OPS),comprising culturing a microorganism, wherein the microorganismcomprising a vector comprising a polynucleotide encoding RhtB(homoserine lactone export transporter) protein variant having an aminoacid sequence of SEQ ID NO: 2, 3, 4, or
 5. 2. The method according toclaim 1, wherein the microorganism further has reduced activity ofendogenous phosphoserine phosphatase (SerB) or enhanced activity ofphosphoserine aminotransferase (SerC) or phosphoglycerate dehydrogenase(SerA) resistant to serine feedback inhibition, or the RhtB proteinvariant.